Goals and Objectives:The overall goal is to understand the biology of cancer cachexia, specifically the role of proinflammatory cytokines and cyclooxygenase activity in tumor-induced weight loss and skeletal muscle wasting. Experiments address the effect of tumor growth on cytokine expression, and the effects of cyclooxygenase inhibitors on intracellular factors (p70, ubiquitin ligases) involved in muscle protein synthesis and degradation, and on spleen cell production of immunoregulatory, proinflammatory (IL-6, TNF) and anti-inflammatory (IL-10)cytokines. Research Significance:Cancer cachexia includes the clinical conditions of anorexia, weight loss, and skeletal muscle wasting. These conditions affect 50-80% of cancer patients, increase symptom distress and negatively impact patient survival. Spleen cell expression of proinflammatory cytokines is increased in weight-losing tumor bearing mice, and muscle cells incubated in the presence of cytokines have increased expression of ubiquitin, ubiquitin ligases, and proteolytic units of the proteasome. Studies conducted in our lab and others have demonstrated that indomethacin and ibuprofen will preserve body weight and muscle mass in tumor-bearing mice and prolong survival in terminally ill cancer patients. The mechanism by which indomethacin or ibuprofen preserve muscle mass is presently unknown, e.g. reduced cytokine expression or reduced activity of the ubiquitin-proteasome pathway. Understanding how these drugs work will increase our understanding of tumor-induced muscle wasting and facilitate the development of targeted interventions to preserve muscle mass and muscle function in patients with cancer cachexia. Methods: Cytokines and other factors associated with weight loss and muscle wasting in tumor-bearing mice are measured in spleen cells and skeletal muscle homogenates of mice using methods of ELISA, western blotting and real time pcr. Tumor cell lines include the colon26 adenocarcinoma and the B16 melanoma. Gene-modified cell lines derived from these parent lines are also used to increase the immune response to the tumor cells, which allows us to profile host production of immunoregulatory (IL-2, IL-12, IL-15) versus proinflammatory (IL-6, TNF) and anti-inflammatory (IL-10) cytokines during the course of tumor growth. Scientific Advances:We have recently demonstrated that growth of the colon26 tumor is associated with reduced muscle levels of phosphorylated p70 s6 kinase (p-p70), a ribosomal kinase involved in muscle protein synthesis. These findings explain in part the reported reduction in muscle protein synthesis in tumor bearing mice and cancer patients. However, indomethacin and ibuprofen, which preserve muscle mass and muscle protein content in mice with the colon26 tumor, have no effect on muscle levels of p-p70. Thus, preservation of muscle mass in the treated animals does not involve this ribosomal kinase. Alternatively, indomethacin does decrease levels of E3 ubiquitin ligases, TNF receptors, and free actin in muscles of treated mice. Paradoxically, we did not find that indomethacin preserved muscle mass or affected levels of TNF receptors or E3 ligases in mice with B16 melanoma tumors. Future Plans:Experiments are underway to use real time pcr to compare the effect of indomethacin and NS398, a COX2 specific inhibotor, on cytokine expression in spleen and skeletal muscle tissue of mice implanted with the colon26 or B16 melanoma. Another series of experiments will profile the expression of proinflammatory and immunoregulatory cytokines early in the course of tumor growth versus later when cachexia is evident (weight loss and muscle wasting). In addition, we will examine the cytokine profile and development of cachexia in mice implanted with gene modified tumor cell lines that have been shown to increase the immune response to tumor growth in vitro.